We have developed and validated a powerful high throughput approach called "Chaperone-Assisted Crystallography" (CAC) that can greatly facilitate solving the most challenging types of structural biology problems. The long-range goal is to develop the CAC platform to a level where its application will have a transforming effect on the structural biology community by making difficult problems routine and "impossible" problems feasible. The CAC methodology is based on the use of synthetically derived antibody fragments as "crystallization chaperones" that specifically and tightly bind to a target protein or RNA entity and thereby promote crystallization and provide phasing information. The technical breakthrough that forms the foundation of the CAC method is an innovative combinatorial library design that employs a "reduced genetic code" to produce highly functional synthetic antibody fragments to an extraordinary broad spectrum of target molecules. Using this approach we have produced chaperones to recalcitrant targets, including membrane proteins and functional RNAs, facilitating crystallization and subsequent structure determination. Major achievements include the structures of the full-length KcsA potassium ion channel and the P4P6 domain of group 1 intron. To further expand the capabilities of the CAC technology, we propose to develop a number of additional enhancements. The 2nd Generation CAC platform will include: i) new "reduced genetic code" chaperone libraries tailored for membrane proteins and nucleic acids, respectively;ii) trapping preferred conformational states of the targeted molecules;iii) targeting chaperones to a specific, predefined region on the target molecule;iv) the ability to produce chaperones to transient macromolecular complexes (e.g. DNA-protein interactions) and stabilize such complexes. v) "co-chaperones" in the form of Fab-binding proteins that can be engineered to introduce anomalous atom types for MAD phasing and alter surface properties, as well as to induce lattice formation. The effectiveness of the new CAC enhancements will be evaluated against a set of five high-impact structural problems that have proven to be totally intractable using traditional approaches. These "model systems" include: 1) HIV 1 Integrase-DNA complex, 2) F-actin tetramer, 3) MerR family of transcription factors, 4) NaChBac Na+ channel, and 5) conformationally locked forms of the 22-adregenic receptor. Experiences gained from working with these sytems will be used to further refine the CAC technology. To make this powerful technology available to the structural biology community, we have established an extramural program where investigators can send us their recalcitrant proteins to be put through the CAC pipeline, or send personnel to our labs to be supervised on producing crystallization chaperones. PUBLIC HEALTH RELEVANCE: We are developing our Chaperone-Assisted Crystallography (CAC) technology to solve the structure of protein systems of critical biomedical importance that have been recalcitrant to crystallization by traditional methods. A key innovation is our ability to rapidly generate designer antibodies that tightly bind the target molecules and promote the formation of high-quality crystals for x-ray crystallographic analysis. The technology will have a broad impact on the structural biology community and the structures determined with it will advance fundamental understanding of cellular functions and provide guidelines for drug development in a variety of diseases.